function of the hd-zip i gene oshox22 in aba-mediated...
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Function of the HD-Zip I gene Oshox22 in ABA-mediated droughtand salt tolerances in rice
Shuxin Zhang • Imran Haider • Wouter Kohlen • Li Jiang •
Harro Bouwmeester • Annemarie H. Meijer • Henriette Schluepmann •
Chun-Ming Liu • Pieter B. F. Ouwerkerk
Received: 24 February 2012 / Accepted: 4 September 2012
� Springer Science+Business Media B.V. 2012
Abstract Oshox22 belongs to the homeodomain-leucine
zipper (HD-Zip) family I of transcription factors, most of which
have unknown functions. Here we show that the expression of
Oshox22 is strongly induced by salt stress, abscisic acid (ABA),
and polyethylene glycol treatment (PEG), and weakly by cold
stress. Trans-activation assays in yeast and transient expression
analyses in rice protoplasts demonstrated that Oshox22 is able
to bind the CAAT(G/C)ATTG element and acts as a tran-
scriptional activator that requires both the HD and Zip domains.
Rice plants homozygous for a T-DNA insertion in the promoter
region of Oshox22 showed reduced Oshox22 expression and
ABA content, decreased sensitivity to ABA, and enhanced
tolerance to drought and salt stresses at the seedling stage. In
contrast, transgenic rice over-expressing Oshox22 showed
increased sensitivity to ABA, increased ABA content, and
decreased drought and salt tolerances. Based on these results,
we conclude that Oshox22 affects ABA biosynthesis and reg-
ulates drought and salt responses through ABA-mediated signal
transduction pathways.
Keywords Rice � Transcription factor � HD-Zip �Drought stress � Regulation � Abiotic stress
Abbreviations
HD-Zip Homeodomain-leucine zipper
ABA Abscisic acid
PEG Polyethylene glycol
GFP Green fluorescent protein
RT Reverse transcription
PCR Polymerase chain reaction
HB Homeobox
Introduction
Drought and salt are major abiotic stresses that cause tre-
mendous yield losses in crops all over the world. Due to
water shortage and less predictable rainfall patterns
resulting from global atmospheric changes, the improve-
ment of stress resistance in crops is now of utmost
importance. Consequently, the genetic basis of drought and
salt resistance is an intensively studied topic. The plant
hormone abscisic acid (ABA) plays a central role in
drought and salt responses through regulating develop-
mental and physiological processes including stomata
closure (Leung and Giraudat 1998; Umezawa et al. 2009;
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-012-9967-1) contains supplementarymaterial, which is available to authorized users.
S. Zhang � L. Jiang � C.-M. Liu (&) � P. B. F. Ouwerkerk (&)
Key Laboratory of Plant Molecular Physiology, Institute
of Botany, Chinese Academy of Sciences,
Beijing 100093, China
e-mail: [email protected]
C.-M. Liu
e-mail: [email protected]
S. Zhang � L. Jiang
Graduate School of Chinese Academy of Sciences, Beijing
100049, China
S. Zhang � A. H. Meijer � P. B. F. Ouwerkerk
Institute of Biology, Leiden University, P.O. BOX 9505, 2300
RA Leiden, The Netherlands
I. Haider � W. Kohlen � H. Bouwmeester
Laboratory of Plant Physiology, Wageningen University,
Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
H. Schluepmann
Department of Molecular Plant Physiology, Utrecht University,
Padualaan 8, 3584 CH Utrecht, The Netherlands
123
Plant Mol Biol
DOI 10.1007/s11103-012-9967-1
Melcher et al. 2010; Huang et al. 2012). Using microarrays,
many genes with expression responding to ABA, drought
and salt treatments have been identified (Seki et al. 2001,
2002; Rabbani et al. 2003; Bray 2004; Yamaguchi-Shino-
zaki and Shinozaki 2006). Additional characterization
identified key components mediating gene-expression
changes in drought responses that include the transcription
factors DREB1A (Kasuga et al. 2004), DREB2A (Sakuma
et al. 2006), SNAC1 (Hu et al. 2006), OsbZIP23 (Xiang
et al. 2008), and DST (Huang et al. 2009). Furthermore, a
number of transcription factors encoded by homeodomain-
leucine zipper (HD-Zip) genes in Arabidopsis, rice and
other plants have been implicated in regulating drought
tolerance through either ABA-dependent or ABA-inde-
pendent pathways (e.g. Soderman et al. 1996, 1999; Gago
et al. 2002; Himmelbach et al. 2002; Deng et al. 2006;
Agalou et al. 2008; Shan et al. 2011). The HD-Zip genes,
however, are an abundant group of transcription factors
that are exclusively found in plants (Ruberti et al. 1991;
Schena and Davis 1992; Aso et al. 1999; Sakakibara et al.
2001; Derelle et al. 2007). HD-Zip proteins, characterized
by a DNA-binding HD and a protein–protein interaction
Zip domain, have been classified into four families (I–IV)
according to their sequence similarities (Ruberti et al.
1991; Morelli and Ruberti 2002). Different members of the
HD-Zip families I and II have been implicated in auxin
signaling and transport (Morelli and Ruberti 2002; Sawa
et al. 2002), vascular development (Scarpella et al. 2000),
and light responses including shade avoidance (Steindler
et al. 1999; Wang et al. 2003). Several other members, such
as Athb-6, Athb-7 and Athb-12 from Arabidopsis
(Soderman et al. 1996, 1999; Lee et al. 2001; Hjellstrom
et al. 2003; Olsson et al. 2004), Hahb-4 from sunflower
(Gago et al. 2002; Dezar et al. 2005a, b; Manavella et al.
2006), and CpHB2, CpHB6 and CpHB7 from Cratero-
stigma plantagineum (Frank et al. 1998; Deng et al. 2002)
are induced by drought and ABA, suggesting a function in
ABA-mediated adaptation to drought stress. In agreement,
inductions of Athb-6, Athb-7 and Athb-12 are abolished in
the ABA-insensitive mutants abi1 and abi2 (Himmelbach
et al. 2002; Olsson et al. 2004).
Like in dicots, a subset of HD-Zip family I and II genes is
regulated by drought in rice (Agalou et al. 2008). Phyloge-
netic analysis places Oshox22, Oshox24, Athb-7 and Athb-12
in the same subgroup (c-clade, Henriksson et al. 2005) of the
HD-Zip family I, and Oshox22 is very likely related to
Oshox24 via an ancient chromosomal duplication (Agalou
et al. 2008). Our previous work showed that Oshox22 is
strongly induced by drought which spurred our interest for
further studies (Agalou et al. 2008). To gain insight into the
function of Oshox22 in drought and salt tolerances, we per-
formed genetic and physiological studies through mutation
and over-expression analyses in rice. Our data showed that
Oshox22 regulates drought and salt stress susceptibility
through an ABA-mediated signaling pathway.
Materials and methods
Plant materials and stress treatments
Drought-tolerant cultivar IRAT 112 (upland tropical
japonica, also named Gajah Mungkur) and drought-sensi-
tive cultivar Nipponbare (lowland japonica) were used for
most studies described in this paper. Zhonghua 11 (lowland
japonica) was used for transgenic analyses. The T-DNA
insertion mutant oshox22-1 in Dongjin (lowland japonica)
background was obtained from the Postech collection in
South Korea.
For hormone treatments, 12 day-old Nipponbare seed-
lings were sprayed with 100 lM ABA, followed by sam-
pling at 0, 1, 3, and 6 h. Alternatively, seedlings at the
same stage were irrigated with 10 % PEG 6,000 or
200 mM NaCl followed by sampling at 0, 1, 3, and 6 h. For
the cold treatment, seedlings were transferred to 4 �C and
sampled after 0, 1, 3, 6, 12, and 24 h. One whole plant was
sampled as one replicate and in total four replicates were
used in each RNA extraction.
For drought treatments, about 40 plants of the oshox22-1
mutant and Oshox22 over-expression lines were grown in
square pots (L26 9 W12 9 H10 cm) filled with a mixture
of sand and soil (1:1) together with wild type, respectively.
We stopped watering when seedlings were 12 days old,
and watering was resumed for 3 days when seedlings were
24 days old, after which the survival rates were calculated.
For salt treatments, the same amount of 12 day-old seed-
lings were irrigated with 150 mM NaCl for 9 days, after
which green leaf rates (green leaf area[30 %) and survival
rates were determined. To measure the water-loss rate
under dehydration conditions, the second leaves from the
top of the plant at the tillering stage (plants were 55 days
old) were cut and exposed to air at room temperature
(approximately 25 �C), and the weight was determined
every 30 min. Every treatment was done in triplicate.
Subcellular localization assay
A full-length cDNA of Oshox22 was amplified from IRAT
112 by RT-PCR using primers Oshox22cdsFW and Osh-
ox22cdsRW (Table S1) based on a full-length cDNA
sequence (GenBank accession AY224440) found in a seed-
derived cDNA expression library (Cooper et al. 2003). The
PCR product was cloned into pCR2.1-TOPO (Invitrogen)
and sequenced. To create a GFP-tagged construct, the full-
length Oshox22 cDNA was excised by EcoRI and ligated
into EcoRI-digested pTH2-BN vector. The cDNA fragment
Plant Mol Biol
123
was inserted to the C-terminus of GFP and expressed using
the Cauliflower mosaic virus (CaMV) 35S promoter. The
construct was transformed into rice protoplasts as previ-
ously described (Chen et al. 2006; Osnato et al. 2010).
Localization of the GFP-tagged Oshox22 protein was
monitored in protoplasts by confocal laser scanning
microscopy (Leica SP5) at 24 h after transformation.
Yeast one-hybrid screens
To assay the activation property, the full-length Oshox22
cDNA PCR product was excised by EcoRI and BamHI,
then ligated into EcoRI/BamHI digested pAS2-1 (Clontech,
GenBank accession U30497), resulting in pAS2-1-
Oshox22. Partial Oshox22 ORFs were also cloned into
pAS2-1 vector, producing different translational fusions
between Oshox22 and the GAL4 BD. Constructs were
sequence-verified before transfer to yeast strain PJ69-4A
(Table S2) for activation screens. All yeast handling and
reporter assays were performed as described before (Meijer
et al. 1998, 2000a; Ouwerkerk and Meijer 2001, 2011).
For the DNA binding assay, a full-length cDNA of
Oshox22 was excised from pAS2-1-Oshox22 with EcoRI
and SalI and then ligated into pRED-ATGa which is rep-
licated via ARS-CEN and maintained via URA3 selection
(unpublished results, P.B.F. Ouwerkerk and A.H. Meijer).
The resulting plasmid pRED-ATG-Oshox22 was assayed in
yeast strains YM4271-4AH1-HIS3 and YM4271-4AH2-
HIS3 which were made using pINT1 as the integrative
vector system (Meijer et al. 1998) which contains the HIS3
reporter gene preceded by AH1 or AH2 tetramer-binding
sites for HD-Zip proteins (Meijer et al. 2000b).
Binary vector constructions and plant transformation
The Oshox22 over-expression construct was made in the
binary vector pC1300intB-35SnosEX (Genbank accession
AY560325) as following: the full-length cDNA of
Oshox22 was excised from pAS2-1-Oshox22 and ligated
into binary vector pC1300intB-35SnosEX, allowing the
gene to be expressed under the control of the CaMV 35S
promoter. We transformed rice (Zhonghua 11) as previ-
ously described (Scarpella et al. 2000) except that the
Agrobacterium tumefaciens strain used was LBA4404.
Calli used for transformation were obtained from germi-
nating seeds according to Rueb et al. (1994). Plantlets were
maintained in culture on half-strength Murashige Skoog
(MS) medium with 10 g/l sucrose until transfer to a
greenhouse (28 �C under a 16 h photoperiod and 85 %
humidity).
Rice protoplast isolation and transient expression
assays
Assays to test DNA binding of Oshox22 involved transient
transformations of protoplasts with effector and reporter
plasmids. The effector plasmid contained the full-length
cDNA of Oshox22 expressed under the control of the
CaMV 35S promoter. The reporter plasmids contained the
putative Oshox22 binding sequences AH1 or AH2 as tet-
ramers upstream of a truncated -90 CaMV 35S promoter
directing GUS gene expression (Meijer et al. 1997). For
protoplast isolation, one hundred rice seeds (Nipponbare)
were grown in 10 cm diameter pots (28 �C, 85 % humid-
ity) in the dark for 12–14 days. Stems and leaves were cut
into *0.5 mm pieces and digested with 25 ml enzyme
solution containing 1.5 % w/v cellulase (Sigma) and 0.3 %
w/v Macerozyme (Sigma) in 50 ml centrifuge tubes. Fur-
ther preparation of protoplasts and transfection with
effector/reporter constructs were as described earlier (Chen
et al. 2006; Osnato et al. 2010). Proteins extraction and
detection of GUS activity were based on Jefferson et al.
(1987). Fluorescence was measured by a Cytofluor 2350
fluorimeter (Millipore).
Northern blot hybridization
Electrophoresis and northern blotting of RNAs were per-
formed as described by Memelink et al. (1994). Baked
blots were pre-hybridized in 1 M NaCl, 1 % SDS, 10 %
dextrane sulphate and 50 lg/ml denatured herring sperm
DNA at 65 �C, washed with 0.1XSSPE, 0.5 % SDS at
42 �C and then autoradiographed. Probes were labeled by
random priming with 32P-dCTP. Equal loading of RNA
samples was verified on the basis of ethidium bromide
staining of ribosomal RNA bands.
Real-time qPCR analysis
Total RNAs from different tissues were pre-treated with
RNase-free DNase I (Takara), according to the manufac-
turer’s instruction. Reverse transcription reaction was
performed with SuperScriptTM III reverse transcriptase
(Invitrogen) following the manufacturer’s instruction.
Primers used for real-time PCR analyses of Oshox22 were
QPCR-22FW and QPCR-22RW (Table S1). qPCR was
performed with Rotor-Gene 3000 Real-time PCR System
using SYBR1 Green to monitor dsDNA synthesis. Relative
expression levels of reporter and target genes were deter-
mined by the Two Standard Curves Relative Quantification
Method using ACTIN1 (Table S1) as the internal control.
Plant Mol Biol
123
Determination of ABA content
Five seeds each of the homozygous T-DNA insertion
mutant oshox22-1 and over-expression lines together with
their corresponding wild type cultivars (Dongjin and
Zhonghua 11 respectively) were surface-sterilized and
germinated in half-strength MS salt solution without
sucrose and grown at 28 �C with a 16/8 h light/dark pho-
toperiod. To determine the ABA level in response to
drought stress, 12 day-old oshox22-1 and over-expression
seedlings were stopped watering and sampled after 10 days
dehydration treatment. Quantification of ABA levels was
performed using LC–MS/MS with five biological replicates
(each 0.2 g of fresh shoot tissue) as described elsewhere,
(Lopez-Raez et al. 2008, 2010). Statistical analyses were
performed using Student’s t test.
Stomata density test
Stomata numbers were counted on the upper epidermis of
the leaf blades of mutant and over-expression lines grown
under normal conditions. Leaf blades were from flag leaves
at the stage that the plants just started to flower. For this
method, oval surfaces spanning 1 9 2 cm were painted
with clear fingernail polish, while avoiding ribbed veins.
After the polish had dried it was peeled off by adding a
tape on the polish and transferred to a microscopy slide.
Finally, the stomata number was counted using DIC
microscopy. Five replicates of every leaf were counted and
from every plant line seven plants were used and all
numbers were converted to number of stomata per square
mm to account for variation in microscopes.
Results
Isolation and sequence analysis of Oshox22
Previous studies showed that the expression of Oshox22 in
rice is strongly induced by drought, and that the induction
is higher in three drought tolerant upland cultivars com-
pared with three lowland cultivars (Agalou et al. 2008). To
elucidate the function of Oshox22, the full-length cDNA of
Oshox22 was amplified from the rice cultivar IRAT 112 by
RT-PCR. The sequence of the amplified cDNA fragment
was identical to that of the cDNA from Nipponbare over
the whole length. Oshox22 (Os04g45810) is located on
chromosome 4 and encodes a protein of 262 amino acids
(AA) including a 61-AA HD domain for DNA binding and
a 43-AA Zip domain for protein–protein interactions.
Expression of Oshox22 under abiotic stress conditions
First, RNA samples from different rice tissues at several
developmental stages were analysed by quantitative RT-PCR
(qRT-PCR) in Nipponbare. The results showed that Oshox22
is ubiquitously expressed, with a lower level in stems and
higher in panicles and seeds (Fig. 1a), which is consistent with
earlier observations (Agalou et al. 2008). We then monitored
the Oshox22 expression under different abiotic stress condi-
tions. As shown in Fig. 1b, Oshox22 was rapidly and strongly
induced by NaCl, PEG and ABA, and weakly induced by low
temperature. These results are in agreement with available
microarray data (http://red.dna.affrc.go.jp/RED/).
Subcellular localization and transcriptional activation
function of Oshox22
To confirm that Oshox22 is a nuclear protein, a GFP-tag-
ged Oshox22 construct under the control of the CaMV 35S
promoter was made, with GFP fused at the C-terminus of
the full-length Oshox22 protein (construct 35S::GFP-
Oshox22). This construct was introduced into rice protop-
lasts by transient transformation and the fusion protein’s
subcellular localization was analysed using confocal laser
scanning microscopy, where the 35S::GFP construct
served as control. As shown in Fig. 1c, the GFP signal was
detected specifically in the nuclei of 35S::GFP-Oshox22
transformed protoplasts, while in the control the GFP sig-
nal was located primarily in the cytoplasm. These data
suggest that Oshox22 is a nuclear-localized protein. In
agreement with this observation, a putative nuclear local-
ization signal sequence (RKRR at the AA 59) was found
near its N-terminus, as analyzed by WoLF PSORT
(http://wolfpsort.seq.cbrc.jp/).
To test whether Oshox22 has any transcription activa-
tion property, Oshox22 was fused to the Gal4p binding
domain (GAL4 BD) and assayed for the ability to induce
expression of a HIS3 gene preceded by Gal4p binding sites.
The result showed that the transformed yeast cells were
able to grow on medium lacking histidine, with up to
10 mM 3-amino-1,2,4-triazole (3-AT), a competitive
inhibitor of His3p enzyme activity (Fig. 2a). In contrast, no
growth was observed of yeast transformed with a control
construct without Oshox22 (pAS2-1), indicating that
Oshox22 has transcriptional activation activity in yeast. To
dissect the activation domain(s), a series of seven truncated
Oshox22 constructs with deletions from either the N- and
C-termini were tested. No activation activity was observed
when either the HD or the Zip domains were deleted
(Fig. 2a), suggesting both domains are required for tran-
scriptional activation in yeast.
Plant Mol Biol
123
Interactions of Oshox22 in yeast and rice
with the CAAT(C/G)ATTG sequence
Previous results showed that HD-Zip family I and II pro-
teins are able to interact with pseudopalindromic AH1
(CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG)
sequences, respectively (Sessa et al. 1993; Meijer et al.
1997, 1998, 2000b; Palena et al. 1999; Johannesson et al.
2001). To test whether Oshox22 interacts with either the
AH1 or AH2 sequences or both, yeast strains containing a
chromosomally integrated HIS3 reporter gene with
upstream AH1 or AH2 tetramers (named 4AH1-HIS3 and
4AH2-HIS3 respectively, Meijer et al. 1998, 2000b) were
used. The Oshox22 ORF was cloned into pRED-ATGa
(named pRED-ATGa-Oshox22), allowing for constitutive
expression of full length Oshox22 protein in yeast without
fusion to an exogenous activation domain. Construct
pRED-ATGa-Oshox22 was transformed into yeast strains
containing constructs 4AH1-HIS3 or 4AH2-HIS3. The
results showed that yeast cells with 4AH2-HIS3
Fig. 1 Expression profiling and
subcellular localization of
Oshox22. a qRT-PCR analyses
of Oshox22 expression in
scutellum-derived calli, root,
shoot, leaf sheath, flag leave,
panicle, stem and mature grain
from rice. b Northern blot
analysis of Oshox22 expressions
in response to ABA (100 lM),
salt (200 mM NaCl), 10 % PEG
6,000 and low-temperature
(4 �C) treatments. c Nuclear
localization of GFP-tagged
Oshox22. The GFP-Oshox22
fusion protein, driven by the
CaMV 35S promoter, was
transiently expressed in rice
protoplasts and visualized by
confocal laser scanning
microscopy. Construct pTH2(Chiu et al. 1996) carrying a
GFP gene driven by CaMV 35S
promoter was used as the
negative control
Plant Mol Biol
123
transformed with pRED-ATGa-Oshox22 grew well on a
medium lacking histidine but containing up to 10 mM
3-AT (Fig. 2b), whereas no growth was observed in yeast
strains with 4AH1-HIS3 or with empty pRED-ATGa vector.
Thus, in yeast, Oshox22 is able to bind AH2, but not AH1,
and can activate reporter gene expression by an intrinsic
activation domain.
To confirm binding of Oshox22 protein to the AH2
sequence transient expression assays were carried out with
effector and reporter plasmids in rice protoplasts. Two
reporter plasmids, 4AH1-90-GUS and 4AH2-90-GUS were
used, in which the AH1 and AH2 tetramers were fused to a
CaMV -90 35S minimal promoter. Construct Pro35S-
Oshox22 with Oshox22 expressed under control of the
Fig. 2 Trans-activation and
DNA-binding specificity of
Oshox22. a Trans-activation
assay in yeast with truncated
Oshox22 protein. Fusion
proteins of the GAL4 DNA-
binding domain and different
fragments of Oshox22 were
examined for their trans-
activation activities in yeast
PJ69-4A. HD, homeodomain;
Zip, leucine zipper. b Trans-
activation assay of Oshox22 in
yeast. Sections 1 and 2, negative
control; sections 3 and 4,
Oshox22 fused to GAL4-BD in
the vector pRED-ATGa was
transformed into yeast strains
YM4271-4AH1-HIS3 and
YM4271-4AH2-HIS3,
respectively. c Interactions of
Oshox22 with the HD-Zip
binding site AH2 (CAAT(C/
G)ATTG) and activation of
reporter gene expression in a
transient expression system
using rice protoplasts. Transient
expression of Oshox22 was
driven by the CaMV 35S
promoter. The Oshox22-OXconstruct was co-transformed
with the reporter constructs
GUSXX-4AH1 or GUSXX-
4AH2. The empty vectors
pRT101 and GUSXX-90 were
used as negative controls
Plant Mol Biol
123
CaMV 35S promoter was used as an effector. As shown in
Fig. 2c, GUS expression in protoplasts co-transformed with
Pro35S-Oshox22 and 4AH2-90-GUS was 3.07 times higher
than those co-transformed with Pro35S-Oshox22 and
4AH1-90-GUS, and 6.11 times higher than those co-
transformed with the empty vector. These data indicate that
Oshox22 is capable to activate transcription of the reporter
gene when upstream HD-Zip binding sites AH1 or AH2 are
present (Fig. 2c). The interaction is less effective at the
AH1 site than that at AH2, which is in contrast to earlier
observations where HD-Zip I proteins mainly interacted
with AH1 and HD-Zip II proteins with AH2 (Sessa et al.
1993; Meijer et al. 1997, 2000b; Palena et al. 1999).
Decreased ABA sensitivity of the oshox22-1 mutant
We searched the publicly available mutant collections and
obtained a putative T-DNA insertion mutant for Oshox22
in rice (POSTECH_C054121, B-11507) (Jeong et al. 2006)
(Fig. 3a). The genomic locus of Oshox22 in this mutant is
shown schematically in Fig. 3b. Alignment of the flanking
sequence tag and the genomic sequence showed that the
T-DNA insertion is located at 978 bp before the transla-
tional start codon of Oshox22. To investigate the function
of Oshox22, an homozygous T-DNA insertion line (named
oshox22-1) was identified, and Southern blot analysis
showed that only one copy of T-DNA was present in
oshox22-1 (Fig. S1). Northern blot analysis showed that in
oshox22-1 the Oshox22 transcript was below detection
level (Fig. 3c). Phenotypic studies showed that oshox22-1
plants exhibited no obvious morphological difference
compared to wild type Dongjin (Fig. 3a). The panicle
shape and the grain number remained unchanged. When
oshox22-1 was backcrossed with Dongjin, all F1 and F2
plants showed the same plant stature in the greenhouse. In
the F2 population, the T-DNA insert segregated in a 3:1
ratio (n = 326), suggesting no embryo or gamete lethality
was involved.
We further analysed the sensitivity of oshox22-1 to
ABA in germination assays. On MS media with 3 and
8 lM ABA, seeds from oshox22-1 showed significantly
higher germination rates (72 and 32 %, respectively) than
those from wild type plants segregated from oshox22-1
backcrosses (40 and 8 % on the medium with 3 and 8 lM
ABA, respectively; Fig. 3d). On control media without
ABA, the germination rates of oshox22-1 and wild type
seeds showed no significant difference. These results sug-
gest that Oshox22 may mediate ABA sensitivity. Next,
oshox22-1 seedlings were tested for ABA sensitivity at the
post-germination stage using media with different
concentrations of ABA. The results showed that shoot
development was less inhibited by exogenous ABA in
oshox22-1, as compared to wild type (Fig. 3e). In sum-
mary, down-regulation of Oshox22 expression in the
Fig. 3 The phenotype and
ABA sensitivity of oshox22-1.
a Morphology of oshox22-1 at
post-anthesis stage. b Schematic
representation of the exon–
intron structure of Oshox22gene and the position of the
T-DNA insert in oshox22-1.
c Oshox22 expression was
down-regulated in oshox22-1.
d Germination rate of oshox22-
1 on MS medium with 0, 3, 5,
and 8 lM ABA for 3 days.
e Relative growth of oshox22-1on medium with 5 lM ABA at
the post-germination stage.
Dongjin represent wild type
plants segregated from a
heterozygous oshox22-1 parent
Plant Mol Biol
123
T-DNA insertion line led to compromised ABA sensitivity
at germination as well as post-germination stages.
Increased ABA sensitivity of transgenic plants over-
expressing Oshox22
For over-expression analysis we expressed Oshox22 under
the control of the CaMV 35S promoter (construct
Oshox22-OX). Northern blot analysis confirmed that
Oshox22 gene expression levels were increased in the
transgenics (Fig. S2). Compared to wild type control
plants, the Oshox22-OX plants exhibited fewer tillers and
decreased height (Fig. 4a). We chose two transgenic lines
(Oshox22-OX-2 and Oshox22-OX-17) for further analyses.
The seed germination rates were reduced 17 % in these two
lines (Fig. 4b). To analyse the sensitivity of the Oshox22-
OX plants to ABA, seeds were germinated on solid MS
media containing either 0, 3, 5 or 8 lM ABA. As shown in
Fig. 4b, germination of seeds from both Oshox22-OX lines
was severely inhibited by all concentrations of ABA used
and the inhibition was much stronger than that observed for
wild type seeds. For instance, the germination rates of the
two Oshox22-OX lines plated on medium with 3 lM ABA
were 40 %, as compared to 80 % on medium without
ABA, whereas in the wild type only a slight reduction
(from 98 to 80 %) was observed. The relatively low
germination rates observed in Oshox22-OX seeds could be
due to an increased ABA sensitivity. We further tested
Oshox22-OX seedlings on media containing 0, 3, 5 or
8 lM ABA and found that the growth of Oshox22-OX
seedlings was inhibited to a greater extent by 5 lM ABA
(Fig. 4c). We therefore concluded that over-expression of
Oshox22 led to increased ABA sensitivity at the germina-
tion as well as post-germination stages.
Functions of Oshox22 in regulating ABA biosynthesis
Next, endogenous ABA levels were measured of the
Oshox22 mis-expression plants grown under control and
drought-stress conditions (Fig. 5). Under normal irrigated
conditions, the ABA content of wild type Dongjin seed-
lings was 9.92 ± 1.06 ng g-1 FW, while in oshox22-1 it
was reduced to 3.86 ± 0.86 ng g-1 FW, an average
reduction of 60 % (Fig. 5a). In wild type Zhonghua 11, the
ABA content was 6.38 ± 0.72 ng g-1 FW, while in over-
expression lines Oshox22-OX-2 and Oshox22-OX-17, the
ABA contents were increased to 7.69 ± 0.42 ng g-1 FW
and 8.35 ± 0.78 ng g-1 FW, respectively (Fig. 5a). Thus,
down-regulation of Oshox22 led to a reduced level of ABA
and over-expression led to increased ABA levels, therefore
we conclude that Oshox22 functions in regulating biosyn-
thesis or degradation of ABA in rice. The differences in
Fig. 4 Phenotype and ABA
sensitivity of Oshox22-OX
plants. a Phenotype of two
independent Oshox22-OX lines
at mature stage. b Germination
rate of seeds from Oshox22-OX
plants on medium with 0, 3, 5,
and 8 lM ABA for 3 days.
c Shoot and root growth of
Oshox22-OX plants on MS
medium with 5 lM ABA at
post-germination stage. ZH11
(Zhonghua 11), wild type
segregated from the T1 line
Plant Mol Biol
123
ABA levels are likely causing changes in sensitivity of
germinating and developing seedlings towards exogenous
applied ABA. Furthermore, we also tested the ABA con-
tents under drought stress conditions. We found that ABA
contents were increased in all plants after drought treat-
ment (Fig. 5b), however, there were differences in the
levels of induction ranging from 9.34 in wild type
Zhonghua 11 to 38.10 in oshox22-1. Although the absolute
ABA level was lower in the mutant, the level of ABA
accumulated 38.31 fold whereas this was 21.39 fold in wild
type Dongjin. In the two Oshox22 overexpression lines the
induction level was 15.04 whereas in control Zhonghua 11
plants this was only 9.34, thus not only the absolute ABA
levels were higher but also the induced levels were higher.
Drought and salt responses in oshox22-1 and Oshox22-
OX plants
To address the function of Oshox22 in drought and salt
responses, we analysed a homozygous oshox22-1 line and
two Oshox22-OX lines for drought and salt tolerances.
When the seedlings were 12 days old (five leaf stage) we
stopped watering for 12 days and then resumed watering
for 3 days to measure the survival rates. Drought treatment
strongly decreased the survival rate of Oshox22-OX plants,
while in oshox22-1 mutants the survival rate was increased,
as compared to their corresponding wild type control
groups (Fig. 6a, b). As shown in Fig. 6c, d, 92 % of the
oshox22-1 seedlings survived, as compared to 34.7 % in
the control group. In contrast, 63 % of the Oshox22-OX
seedlings survived, as compared to 87 % in their control
group. From these data we conclude that mutation of
Oshox22 led to increased drought tolerance, while over-
expression of Oshox22 led to decreased drought tolerance.
Furthermore, we measured water-loss rates in leaves from
oshox22-1 and Oshox22-OX plants during the dehydration
process. Leaves from oshox22-1 had significantly lower rates
(P \ 0.05) of water-loss than control plants (Fig. 6e). In
contrast, Oshox22-OX plants showed no significant differ-
ence in water-loss rate as compared to the control (Fig. 6f).
Because stomata density is a critical factor in drought stress,
we determined the stomata density in leaf blades of oshox22-
1 plants at flowering stage, Oshox22-OX, and their corre-
sponding wild type control plants. The oshox22-1 leaves
showed 17.8 % reduction in stomata density, but stomata
density remained unchanged in Oshox22-OX, as compared
to leaves from wild type plants (Fig. 7). Therefore, the
enhanced drought tolerance in oshox22-1 may partly result
from decreased stomata density in leaves.
To evaluate salt tolerance in oshox22-1 and Oshox22-
OX plants, 12-day-old seedlings grown in hydroponic
culture were transferred to a 150 mM NaCl solution for
9 days and then the green leaf area was measured. The
oshox22-1 plants had significantly more green leaf area
(80 %) than control plants (36 %; Fig. 8). In contrast,
Oshox22-OX plants had a reduced green leaf area (65 %)
compared with the control (86 %; Fig. 8). These results
suggest that in rice, down-regulation of Oshox22 improved
salt tolerance, whilst over-expression of Oshox22 led to
reduced salt tolerance. These data further suggest that
Oshox22 functions as a negative regulator in drought and
salt tolerance in rice.
Discussion
In rice and Arabidopsis, about 50 % of all homeobox genes
(Chan et al. 1998; Jain et al. 2008; Mukherjee et al. 2009)
belong to the HD-Zip family. Although some members
have been implicated in the regulation of development and
stress responses, the functions of most HD-Zip genes are
still unknown. Based on mutant and over-expression
analyses, we here propose a function for the rice HD-Zip
Fig. 5 Effects of Oshox22 mis-expression on ABA levels. ABA
levels in oshox22-1 and Oshox22-OX plants and wild type cultivars
Dongjin and Zhonghua 11. a ABA levels under normal condition.
b ABA levels after drought treatment. After drought treatment, the
ABA levels increased with a factor 21.39, 38.10, 9.34, 15.04 and
15.04 for oshox22-1, Dongjin, Zhonghua 11, Oshox22-OX-2 and
Oshox22-OX-17, respectively
Plant Mol Biol
123
family I gene Oshox22 in regulating ABA biosynthesis and
ABA-mediated drought and salt tolerances in rice.
In this study, we used a GFP-tagged fusion construct to
demonstrate that Oshox22 is a nuclear-localized protein,
which is consistent with a function as transcription factor.
The DNA-binding and activation properties were analysed in
yeast one-hybrid experiments, showing specific binding to a
known HD-Zip target sequence and activation of reporter
gene expression without the requirement of exogenous
activation domains. Further functional analysis in yeast
showed that both HD and Zip domains are required for the
trans-activation. The function of Oshox22 as a transcrip-
tional activator was further confirmed with transient assays
in rice protoplasts using a GUS reporter gene. Sessa et al.
(1993, 1997) propose that AH1 (CAAT(A/T)ATTG) and
AH2 (CAAT(C/G)ATTG) act as consensus binding sites for
Fig. 6 Drought tolerance of
oshox22-1 and Oshox22-OX
plants. a Seedlings of oshox22-1and Oshox22-OX before
applying the drought-stress.
b After drought stress treatment.
c, d, Survival rates of oshox22-1and Oshox22-OX plants after
application of drought stresses.
e Water-loss rates in leaves
from oshox22-1 and
corresponding wild type plants.
f Water-loss rate in leaves from
Oshox22-OX and corresponding
wild type plants
Plant Mol Biol
123
HD-Zip I and HD-Zip II proteins, respectively. Apparently,
Oshox1 to Oshox7 follow these rules (Meijer et al. 1997,
1998, 2000b). Athb-7 and Athb-12, however, do not seem to
bind to either of these consensus sequences and may have
totally different binding preferences (Himmelbach et al.
2002; Deng et al. 2006). Our data show that Oshox22 is able
to activate gene expression via both AH1 and AH2, but more
effectively via AH2 than AH1. Taken together, our data from
the yeast and protoplast experiments support the function of
Oshox22 as a transcriptional activator, which is typical of
HD-Zip I family transcription factors (Ohgishi et al. 2001;
Meijer et al. 2000b).
Similar to closely related HD-Zip factors such as Athb-
6, Athb-7 and Athb-12, our previous (Agalou et al. 2008)
and current work shows that expression of Oshox22 is
responsive to drought, salinity and ABA treatments, sug-
gesting its role in regulating stress tolerance. In Arabid-
opsis, the HD-Zip I protein Athb-6 has been shown to
interact with ABI1, a protein phosphatase 2C (Himmelbach
et al. 2002). ABI1 is involved in various responses towards
ABA including stomata closure, seed dormancy and veg-
etative growth and thus represents a key component in
ABA signaling (Leung et al. 1997; Leung and Giraudat
1998). For Athb-7 and Athb-12, it has been found that their
expression is down-regulated in abi1 mutants (Olsson et al.
2004), which further supports the interaction between ABA
signaling and these HD-Zip genes. To perform functional
analyses, we generated Oshox22 over-expression lines and
obtained an oshox22 mutant that contains a T-DNA
insertion in the 50 upstream sequence of Oshox22 and
displays strongly down-regulated expression of this gene.
Under normal greenhouse conditions, oshox22-1 plants did
not show any visible difference from wild type. However,
the endogenous ABA levels in oshox22-1 seedlings were
60 % lower than those of the wild type Dongjin plants.
When grown on medium with ABA, oshox22-1 seeds
showed a higher germination rate than the wild type. On
the other hand, rice plants over-expressing Oshox22
showed a decreased germination rate on medium supplied
with ABA suggesting Oshox22 is involved in an ABA-
regulated seed germination process. It should be noted that
the over-expression and mutation phenotypes were ana-
lysed in different genetic backgrounds. Zhonghua 11 is an
important lowland rice cultivar in Chinese agriculture
which is easy to transform using A. tumefaciens and
therefore this is the preferred model for transgenesis in our
laboratory. As a mutant allele of Oshox22 was not present
in the Zhonghua 11 RMD T-DNA collection (Zhang et al.
2006), we analysed a mutant allele of Oshox22 in Dongjin,
which is also a lowland Japonica rice cultivar. Since ABA
sensitivity was decreased upon Oshox22 mutation in
Dongjin background and increased upon over-expression in
Zhonghua 11 background, our data suggest that the func-
tion of Oshox22 between these cultivars is conserved.
These results of Oshox22 effects on ABA sensitivity are
only partly consistent with those obtained in Arabidopsis
with Athb-7 and Athb-12, in spite of the fact that the latter
are in the same HD-Zip I subgroup (c-clade) as Oshox22
(Olsson et al. 2004; Henriksson et al. 2005; Agalou et al.
2008). Furthermore, over-expression of CpHB-7 isolated
from C. plantagineum in Arabidopsis resulted in increased
germination on ABA and thus reduced sensitivity towards
ABA (Deng et al. 2006). Plants over-expressing Athb-6 are
also less sensitive to ABA on germination (Himmelbach
et al. 2002). Thus, different HD-Zip proteins may be
involved in different ABA-mediated signaling pathways
and differences of closely related genes in the different
species suggest that the signaling pathway may have
evolved rapidly.
Although Oshox22 is induced by ABA, constitutive
over-expression of Oshox22 in Zhonghua 11, led to an
increased ABA level but compromised drought tolerance.
Fig. 7 Stomata counting in leaves from oshox22-1 and Oshox22-OX
plants. a The number of stomata on oshox22-1 leaves compared to
wild type (P \ 0.01, n = 10). b The number of stomata on two
independent Oshox22-OX lines compared to wild type (P [ 0.05,
n = 10). Dongjin in this context is the wild type segregated from
heterozygous oshox22-1/Oshox22 line; ZH11 (Zhonghua 11), wild
type segregated from an Oshox22-OX T1 line
Plant Mol Biol
123
In addition to decreasing ABA sensitivity, however, the
Oshox22 mutation in Dongjin resulted in an increased
tolerance towards drought, which correlated with reduced
water-loss efficiencies of mutant seedlings. As numbers of
stomata were decreased in oshox22-1 mutants, regulation
of stomata density by Oshox22 is a possible mechanism
underlying enhanced drought tolerance in these mutants.
However, a change in stomata density was not found in the
Oshox22-OX lines although these lines were less drought
tolerant. Therefore, most likely stomata density is not the
only factor determining drought tolerance under our
experimental conditions. We conclude that Oshox22 neg-
atively regulates drought tolerance in rice. In wheat a
similar situation is described where the level of ABA is
inversely correlated to the level of drought tolerance. In
drought-sensitive cultivars, drought treatment leads to
enhanced expression of ABA biosynthesis genes in anthers
and ABA accumulation in spikes, while in drought-tolerant
wheat the treatment leads to accumulation of lower levels
of ABA, which correlates with lower expression of ABA
biosynthesis genes and higher level of expression of ABA
catabolic genes (e.g. ABA 80-hydroxylase). Furthermore,
wheat TaABA80 OH1 deletion lines that accumulate higher
levels of ABA in spikes are drought sensitive (Ji et al.
2011). We analysed ABA levels in oshox22-1 mutants and
the two over-expression lines as well as their respective
wild type backgrounds. We found that mis-expression of
Oshox22 affected the absolute levels of ABA. However,
despite difference in induction levels, in all plants ABA
levels increased strongly in response to drought, which is in
line with the well-known function of this hormone in
responses to stress signals including drought and salinity.
Higher levels of ABA are due to induction of ABA bio-
synthesis genes and in turn ABA reprograms plant cells to
withstand and survive adverse environmental conditions
(reviewed by Leung and Giraudat 1998; Umezawa et al.
2009; Melcher et al. 2010; Huang et al. 2012). We
hypothesize that Oshox22 may plays a role in ABA bio-
synthesis or degradation and that a lower endogenous ABA
level in oshox22-1 plants and higher level of ABA,
respectively correlate inversely with their drought toler-
ance. Being a transcription factor, Oshox22 may affect
Fig. 8 Examinations of salt
tolerance of oshox22-1 and
Oshox22-OX plants.
a Seedlings of oshox22-1 and
Oshox22-OX before salt
treatments. b Plants treated with
150 mM NaCl for 9 days. c,
d Green leaf rates for oshox22-
1, Oshox22-OX and their wild
type plants after salt treatments
Plant Mol Biol
123
ABA level by directly regulating genes involved in bio-
synthesis or degradation of ABA.
We propose that Oshox22 functions as a negative reg-
ulator in drought and salt tolerance similar to OsABI5,
which is a bZIP transcription factor, and is inducible by
ABA and high salinity (Zou et al. 2008). Transgenic rice
plants over-expressing OsABI5 were sensitive to ABA and
to high-salinity stress as well as to PEG treatment. Similar
as with the oshox22-1 mutant, down-regulation of OsABI5
in plants using an RNAi approach exhibited increased
tolerance to salt as well as to PEG treatment which is a
stress condition we did not check for Oshox22 (Zou et al.
2007, 2008). It seems that several regulators exist that are
able to control both drought and salt tolerances. Other
examples are the transcription factors OsbZIP23 (Xiang
et al. 2008) and DST (Huang et al. 2009). However, there
are also differences with Oshox22. Like with Oshox22,
both factors are nuclear-localized transcriptional activators
and down-regulation of OsbZIP23 expression results in
decreased ABA sensitivity but tolerance towards salt and
drought stress is decreased and over-expression results in
an increase of tolerance which is opposite to the results
with Oshox22. Microarray experiments with OsbZIP23
mis-expression plants identified sets of genes regulated by
OsbZIP23 amongst which many genes have known func-
tions in stress tolerance (Xiang et al. 2008). Interestingly,
the microarray dataset shows that Oshox22 as well as
Oshox24 which is on a duplicated chromosome segment
(Agalou et al. 2008), are higher expressed in the OsbZIP23
over-expressor indicating that this gene probably acts
upstream of the two HD-Zip I genes. We expect that in turn
Oshox22 and Oshox24 also have similar functions in
controlling other sets of stress tolerance genes. Future
experiments can involve similar experiments for down-
stream target genes in order to explain how Oshox22 reg-
ulates drought and salt tolerance and ABA biosynthesis.
Taken together, down-regulation of Oshox22 expression
by T-DNA insertion led to plants with reduced levels of
ABA and enhanced tolerance towards drought and salt
stresses, while over-expression of the gene increased ABA
content and decreased drought and salt tolerances. These
results support the conclusion that Oshox22 acts as a
negative regulator in stress responses. Since reporter gene
studies in yeast and rice cells suggested that Oshox22 acts
as a transcriptional activator, its function as a negative
regulator in stress responses might be explained via acti-
vation of other repressors. The fact that oshox22-1 plants
showed no significant reduction in yield makes it a
potential candidate for improving stress tolerance in rice.
Further research is needed to identify allelic variation
related to altered levels of Oshox22 expression for rice
breeding.
Acknowledgments We acknowledge the support from projects
CEDROME (INCO-CT-2005-015468) for PBFO and CML, the
National Natural Science Foundation of China (30821007) and the
CAS/SAFEA International Partnership Program for Creative
Research Teams (20090491019) for SZ and CML, TF-STRESS
(QLK3-CT-2000-00328) for AHM, RNA Seed from the Royal
Netherlands Academy of Arts and Sciences (KNAW-CEP
08CDP036) for SZ, PBFO and HS, from the Higher Education
Commission (HEC) Pakistan to IH and the Netherlands Organization
for Scientific Research (NWO; VICI-grant to HB). We thank Francel
Verstappen for his technical support at WUR in The Netherlands.
References
Agalou A, Purwantomo S, Overnas E, Johannesson H, Zhu X, Estiati
A, de Kam RJ, Engstrom P, Slamet-Loedin IH, Zhu Z, Wang M,
Xiong L, Meijer AH, Ouwerkerk PBF (2008) A genome-wide
survey of HD-Zip genes in rice and analysis of drought-
responsive family members. Plant Mol Biol 66:87–103
Aso K, Kato M, Banks JA, Hasebe M (1999) Characterization of
homeodomain-leucine zipper genes in the fern Ceratopterisrichardii and the evolution of the homeodomain-leucine zipper
gene family in vascular plants. Mol Biol Evol 16:544–552
Bray EA (2004) Genes commonly regulated by water-deficit stress in
Arabidopsis thaliana. J Exp Bot 55:2331–2341
Chan RL, Gago GM, Palena CM, Gonzalez DH (1998) Homeoboxes
in plant development. Biochim Biophys Acta 1442:1–19
Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL
(2006) A highly efficient transient protoplast system for
analyzing defence gene expression and protein–protein interac-
tions in rice. Mol Plant Pathol 7:417–427
Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996)
Engineered GFP as a vital reporter in plants. Curr Biol
6:325–330
Cooper B, Clarke JD, Budworth P, Kreps J, Hutchison D, Park S,
Guimil S, Dunn M, Luginbuhl P, Ellero C, Goff SA, Glazebrook
J (2003) A network of rice genes associated with stress response
and seed development. Proc Natl Acad Sci USA 100:4945–4950
Deng X, Phillips J, Meijer AH, Salamini F, Bartels D (2002)
Characterization of five novel dehydration-responsive homeo-
domain leucine zipper genes from the resurrection plant
Craterostigma plantagineum. Plant Mol Biol 49:601–610
Deng X, Phillips J, Brautigam A, Engstrom P, Johannesson H,
Ouwerkerk PBF, Ruberti I, Salinas J, Vera P, Iannacone R,
Meijer AH, Bartels D (2006) A homeodomain leucine zipper
gene from Craterostigma plantagineum regulates abscisic acid
responsive gene expression and physiological responses. Plant
Mol Biol 61:469–489
Derelle R, Lopez P, Le Guyader H, Manuel M (2007) Homeodomain
proteins belong to the ancestral molecular toolkit of eukaryotes.
Evol Dev 9:212–219
Dezar CA, Gago GM, Gonzalez DH, Chan RL (2005a) HAHB-4, a
sunflower homeobox-leucine zipper gene, confers drought
tolerance to Arabidopsis thaliana plants. Transgenic Res
14:429–440
Dezar CA, Fedrigo GV, Chan RL (2005b) The promoter of the
sunflower HD-Zip protein gene HAHB4 directs tissue-specific
expression and is inducible by water stress, high salt concentra-
tions and ABA. Plant Sci 169:447–459
Frank W, Phillips J, Salamini F, Bartels D (1998) Two dehydration
inducible transcripts from the resurrection plant Craterostigmaplantagineum encode interacting homeodomain-leucine zipper
proteins. Plant J 15:413–421
Plant Mol Biol
123
Gago GM, Almoguera C, Jordano J, Gonzales DH, Chan RL (2002)
Hahb-4, a homeobox-leucine zipper gene potentially involved in
abscisic acid-dependent responses to water stress in sunflower.
Plant, Cell Environ 25:633–640
Henriksson E, Olsson ASB, Johannesson H, Johansson H, Hanson J,
Engstrom P, Soderman E (2005) Homeodomain leucine zipper
class I genes in Arabidopsis. Expression patterns and phyloge-
netic relationships. Plant Physiol 139:509–518
Himmelbach A, Hoffmann T, Leube M, Hohner B, Grill E (2002)
Homeodomain protein Athb6 is a target of the protein phospha-
tase ABI1 and regulates hormone responses in Arabidopsis.
EMBO J 21:3029–3038
Hjellstrom M, Olsson ASB, Engstrom P, Soderman EM (2003)
Constitutive expression of the water deficit-inducible homeobox
gene Athb7 in transgenic Arabidopsis causes a suppression of
stem elongation growth. Plant, Cell Environ 26:1127–1136
Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong LZ (2006)
Overexpressing a NAM, ATAF, and CUC (NAC) transcription
factor enhances drought resistance and salt tolerance in rice. Proc
Natl Acad Sci USA 103:12987–12992
Huang XY, Chao DY, Gao JP, Zhu MZ, Shi M, Lin HX (2009) A
previously unknown zinc finger protein, DST, regulates drought
and salt tolerance in rice via stomatal aperture control. Genes
Dev 23:1805–1817
Huang G-T, Ma S-L, Bai L-P, Zhang L, Ma H, Jia P, Liu J, Zhong M,
Guo Z-F (2012) Signal transduction during cold, salt, and
drought stresses in plants. Mol Biol Rep 39:969–987
Jain M, Tyagi AK, Khurana JP (2008) Genome-wide identification,
classification, evolutionary expansion and expression analyses of
homeobox genes in rice. FEBS J 275:2845–2861
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-
glucuronidase as a sensitive and versatile gene fusion marker in
higher plants. EMBO J 6:3901–3907
Jeong DH, An S, Park S, Kang HG, Park GG, Kim SR, Sim J, Kim
YO, Kim MK, Kim SR, Kim J, Shin M, Jung M, An G (2006)
Generation of a flanking sequence-tag database for activation-
tagging lines in japonica rice. Plant J 45:123–132
Ji X, Dong B, Shiran B, Talbot MJ, Edlington JE, Hughes T, White RG,
Gubler F, Dolferus R (2011) Control of abscisic acid catabolism
and abscisic acid homeostasis is important for reproductive stage
stress tolerance in cereals. Plant Physiol 156:647–662
Johannesson H, Wang Y, Engstrom P (2001) DNA-binding and
dimerisation preferences of Arabidopsis homeodomain-leucine
zipper transcription factors in vitro. Plant Mol Biol 45:63–73
Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2004) A
combination of the Arabidopsis DREB1A gene and stress-
inducible rd29A promoter improved drought- and low-temper-
ature stress tolerance in tobacco by gene transfer. Plant Cell
Physiol 45:346–350
Lee YH, Oh HS, Cheon CI, Hwang IT, Kim YJ, Chun JY (2001)
Structure and expression of the Arabidopsis thaliana homeobox
gene Athb-12. Biochem Biophys Res Commun 284:133–141
Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu
Rev Plant Physiol Plant Mol Biol 49:199–222
Leung J, Merlot S, Giraudat J (1997) The Arabidopsis ABSCISICACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homolo-
gous protein phosphatases 2C involved in abscisic acid signal
transduction. Plant cell 9:759–771
Lopez-Raez JA, Charnikhova T, Gomez-Roldan V, Matusova R,
Kohlen W, De Vos R, Verstappen F, Puech-Pages V, Becard G,
Mulder P, Bouwmeester H (2008) Tomato strigolactones are
derived from carotenoids and their biosynthesis is promoted by
phosphate starvation. New Phytol 178:863–874
Lopez-Raez JA, Kohlen W, Charnikhova T, Mulder P, Undas AK,
Sergeant MJ, Verstappen F, Bugg TDH, Thompson AJ, Ruyter-
Spira C (2010) Does abscisic acid affect strigolactone biosyn-
thesis? New Phytol 187:343–354
Manavella PA, Arce AL, Dezar CA, Bitton F, Renou FP, Crespi M,
Chan RL (2006) Cross-talk between ethylene and drought
signaling pathways is mediated by the sunflower Hahb-4transcription factor. Plant J 48:125–137
Meijer AH, Scarpella E, van Dijk EL, Qin L, Taal AJ, Rueb S,
Harrington SE, McCouch SR, Schilperoort RA, Hoge JHC
(1997) Transcriptional repression by Oshox1, a novel homeodo-
main leucine zipper protein from rice. Plant J 11:263–276
Meijer AH, Ouwerkerk PBF, Hoge JHC (1998) Vectors for
transcription factor isolation and target gene identification by
means of genetic selection in yeast. Yeast 14:1407–1416
Meijer AH, Schouten J, Ouwerkerk PBF, Hoge JHC (2000a) Yeast as
versatile tool in transcription factor research. In: Gelvin SB,
Schilperoort RA (eds) Plant molecular biology manual (chap E3,
2nd edn, suppl IV). Kluwer, Dordrecht, pp 1–28
Meijer AH, de Kam RJ, d’Ehrfurth I, Shen W, Hoge JHC (2000b)
HD-Zip proteins of families I and II from rice: interactions and
functional properties. Mol Gen Genet 263:12–21
Melcher K, Zhou XE, Xu HE (2010) Thirsty plants and beyond:
structural mechanisms of abscisic acid perception and signaling.
Curr Opin Struct Biol 20:722–729
Memelink J, Swords KMM, Staehelin LA, Hoge JHC (1994)
Southern, northern and western blot analysis. In: Gelvin SB,
Schilperoort RA (eds) Plant molecular biology manual. Kluwer,
Dordrecht, pp F1–F23
Morelli G, Ruberti I (2002) Light and shade in the photocontrol of
Arabidopsis growth. Trends Plant Sci 7:399–404
Mukherjee K, Brocchieri L, Burglin TR (2009) A comprehensive
classification and revolutionary analysis of plant homeobox
genes. Mol Biol Evol 26:2775–2794
Ohgishi M, Oka A, Morelli G, Ruberti I, Aoyama T (2001) Negative
autoregulation of the Arabidopsis homeobox gene Athb-2. Plant
J 25:389–398
Olsson ASB, Engstrom P, Soderman E (2004) The homeobox genes
Athb12 and Athb7 encode potential regulators of growth in response
to water deficit in Arabidopsis. Plant Mol Biol 55:663–677
Osnato M, Stile MR, Wang Y, Meynard D, Curiale S, Guiderdoni E,
Liu Y, Horner DS, Ouwerkerk PBF, Pozzi C, Muller KI,
Salamini F, Rossini L (2010) Cross talk between the KNOX and
ethylene pathways is mediated by intron-binding transcription
factors in barley. Plant Physiol 154:1616–1632
Ouwerkerk PBF, Meijer AH (2001) Yeast one-hybrid screening for
DNA-protein interactions. Curr Prot Mol Biol 12.12.1–12.12.22
Ouwerkerk PBF, Meijer AH (2011) Yeast one-hybrid screens for
detection of transcription factor DNA interactions. Methods Mol
Biol 678:211–227
Palena CM, Gonzalez DH, Chan RL (1999) A monomer-dimer
equilibrium modulates the interaction of the sunflower homeo-
domain leucine-zipper protein HaHB-4 with DNA. Biochem J
341:81–87
Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y,
Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K
(2003) Monitoring expression profiles of rice genes under cold,
drought, and high-salinity stresses and abscisic acid application
using cDNA microarray and RNA gel-blot analyses. Plant
Physiol 133:1755–1767
Ruberti I, Sessa G, Lucchetti S, Morelli G (1991) A novel class of
plant proteins containing a homeodomain with a closely linked
leucine zipper motif. EMBO J 10:1787–1791
Rueb S, Leneman M, Schilperoort RA, Hensgens LAM (1994)
Efficient plant regeneration through somatic embryogenesis from
callus induced on mature rice embryos (Oryza sativa L.). Plant
Cell Tiss Org Cult 36:259–264
Plant Mol Biol
123
Sakakibara K, Nishiyama T, Kato M, Hasebe M (2001) Isolation of
homeodomain-leucine zipper genes from the moss Physcomit-rella patens and the evolution of homeodomain-leucine zipper
genes in land plants. Mol Biol Evol 18:491–502
Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K,
Yamaguchi-Shinozaki K (2006) Functional analysis of an
Arabidopsis transcription factor, DREB2A, involved in
drought-responsive gene expression. Plant Cell 18:1292–1309
Sawa S, Ohgishi M, Goda H, Higuchi K, Shimada Y, Yoshida S,
Koshiba T (2002) The HAT2 gene, a member of the HD-Zip
gene family, isolated as an auxin inducible gene by DNA
microarray screening, affects auxin response in Arabidopsis.
Plant J 32:1011–1022
Scarpella E, Rueb S, Boot KJM, Hoge JHC, Meijer AH (2000) A role
for the rice homeobox gene Oshox1 in provascular cell fate
commitment. Development 127:3655–3669
Schena M, Davis RW (1992) HD-Zip protein members of Arabidopsis
homeodomain protein superfamily. Proc Natl Acad Sci USA
89:3894–3898
Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K,
Carnici P, Hayashizaki Y, Shinozaki K (2001) Monitoring the
expression pattern of 1300 Arabidopsis genes under drought and
cold stresses by using a full-length cDNA microarray. Plant Cell
13:61–72
Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A,
Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T,
Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y,
Shinozaki K (2002) Monitoring the expression profiles of 7000
Arabidopsis genes under drought, cold, and high-salinity stresses
using a full-length cDNA microarray. Plant J 31:279–292
Sessa G, Morelli G, Ruberti I (1993) The ATHB-1 and -2 HD-Zip
domains homodimerize forming complexes of different DNA-
binding specificities. EMBO J 12:3507–3517
Sessa G, Morelli G, Ruberti I (1997) DNA-binding specificity of the
homeodomain leucine zipper domain. J Mol Biol 274:303–309
Shan H, Chen S, Jiang J, Chen F, Chen Y, Gu C, Li P, Song A, Zhu X
Gao H Zhou G, Li T, Yang X (2011, in press) Heterologous
expression of the Chrysanthemum R2R3-MYB transcription
factor CmMYB2 enhances drought and salinity tolerance,
increases hypersensitivity to ABA and delays flowering in
Arabidopsis thaliana. Mol Biotech 51:160–173
Soderman E, Mattsson J, Engstrom P (1996) The Arabidopsishomeobox gene Athb-7 is induced by water deficit and by
abscisic acid. Plant J 10:375–381
Soderman E, Hjellstrom M, Fahleson J, Engstrom P (1999) The HD-
Zip gene Athb6 in Arabidopsis is expressed in developing leaves,
roots and carpels and up-regulated by water deficit conditions.
Plant Mol Biol 40:1073–1083
Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T,
Morelli G, Ruberti I (1999) Shade avoidance responses are
mediated by the Athb-2 HD-Zip protein, a negative regulator of
gene expression. Development 126:4235–4245
Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F,
Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K
(2009) Type 2C protein phosphatases directly regulate abscisic
acid-activated protein kinases in Arabidopsis. Proc Natl Acad
Sci USA 106:17588–17593
Wang Y, Henriksson E, Soderman E, Henriksson NK, Sundberg E,
Engstrom P (2003) The Arabidopsis homeobox gene, ATHB16,
regulates leaf development and the sensitivity to photoperiod in
Arabidopsis. Dev Biol 264:228–239
Xiang Y, Tang N, Du H, Ye H, Xiong LZ (2008) Characterization of
OsbZIP23 as a key player of the basic leucine zipper transcrip-
tion factor family for conferring abscisic acid sensitivity and
salinity and drought tolerance in rice. Plant Physiol
148:1938–1952
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regu-
latory networks in cellular responses and tolerance to dehydra-
tion and cold stresses. Annu Rev Plant Biol 57:781–803
Zhang J, Li C, Wu C, Xiong L, Chen G, Zhang Q, Wang S (2006)
RMD: a rice mutant database for functional analysis of the rice
genome. Nucl Acid Res 34:D745–D748
Zou MJ, Guan YC, Ren HB, Zhang F, Chen F (2007) Characterization
of alternative splicing products of bZIP transcription factors
OsABI5. Biochem Biophys Res Commun 360:307–313
Zou MJ, Guan YC, Ren HB, Zhang F, Chen F (2008) A bZIP
transcription factor, OsABI5, is involved in rice fertility and
stress tolerance. Plant Mol Biol 66:675–683
Plant Mol Biol
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